Benchmarking of single-virus genomics: a new tool for uncovering the virosphere

Metagenomics and single-cell genomics have enabled the discovery of relevant uncultured microbes. Recently, single-virus genomics (SVG), although still in an incipient stage, has opened new avenues in viral ecology by allowing the sequencing of one single virus at a time. The investigation of methodological alternatives and optimization of existing procedures for SVG is paramount to deliver high-quality genomic data. We report a sequencing dataset of viral single-amplified genomes (vSAGs) from cultured and uncultured viruses obtained by applying different conditions in each SVG step, from viral preservation and novel whole-genome amplification (WGA) to sequencing platforms and genome assembly. Sequencing data showed that cryopreservation and mild fixation were compatible with WGA, although fresh samples delivered better genome quality data. The novel TruPrime WGA, based on primase-polymerase features, and WGA-X employing a thermostable phi29 polymerase, were proven to be with sufficient sensitivity in SVG. The Oxford Nanopore (ON) sequencing platform did not provide a significant improvement of vSAG assembly compared to Illumina alone. Finally, the SPAdes assembler performed the best. Overall, our results represent a valuable genomic dataset that will help to standardized and advance new tools in viral ecology.     

Mannose receptor (MR) engagement by mesothelin GPI anchor polarizes tumor-associated macrophages and is blocked by anti-MR human recombinant antibody

Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206(low) macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.     

Determinants of inapparent and symptomatic dengue infection in a prospective study of primary school children in Kamphaeng Phet, Thailand

Background

Dengue viruses are a major cause of morbidity in tropical and subtropical regions of the world. Inapparent dengue is an important component of the overall burden of dengue infection. It provides a source of infection for mosquito transmission during the course of an epidemic, yet by definition is undetected by health care providers. Previous studies of inapparent or subclinical infection have reported varying ratios of symptomatic to inapparent dengue infection.

Methodology/Principal Findings

In a prospective study of school children in Northern Thailand, we describe the spatial and temporal variation of the symptomatic to inapparent (S:I) dengue illness ratio. Our findings indicate that there is a wide fluctuation in this ratio between and among schools in a given year and within schools over several dengue seasons. The most important determinants of this S:I ratio for a given school were the incidence of dengue infection in a given year and the incidence of infection in the preceding year. We found no association between the S:I ratio and age in our population.

Conclusions/Significance

Our findings point to an important aspect of virus-host interactions at either a population or individual level possibly due to an effect of heterotypic cross-reactive immunity to reduce dengue disease severity. These findings have important implications for future dengue vaccines.     

Preexisting Japanese encephalitis virus neutralizing antibodies and increased symptomatic dengue illness in a school-based cohort in Thailand

Background

Dengue viruses (DENVs) and Japanese encephalitis virus (JEV) have significant cross-reactivity in serological assays; the clinical implications of this remain undefined. An improved understanding of whether and how JEV immunity modulates the clinical outcome of DENV infection is important as large-scale DENV vaccine trials will commence in areas where JEV is co-endemic and/or JEV immunization is routine.

Methods and Findings

The association between preexisting JEV neutralizing antibodies (NAbs) and the clinical severity of DENV infection was evaluated in a prospective school-based cohort in Thailand that captured asymptomatic, non-hospitalized, and hospitalized DENV infections. Covariates considered included age, baseline DENV antibody status, school of attendance, epidemic year, and infecting DENV serotype. 942 children experienced at least one DENV infection between 1998 and 2002, out of 3,687 children who were enrolled for at least one full year. In crude analysis, the presence of JEV NAbs was associated with an increased occurrence of symptomatic versus asymptomatic infection (odds ratio [OR] = 1.55, 95% CI: 1.08–2.23) but not hospitalized illness or dengue hemorrhagic fever (DHF). The association was strongest in children with negative DENV serology (DENV-naive) (OR = 2.75, 95% CI: 1.12–6.72), for whom the presence of JEV NAbs was also associated with a symptomatic illness of longer duration (5.4 days for JEV NAb+ versus 2.6 days for JEV NAb-, p = 0.048). JEV NAbs were associated with increased DHF in younger children with multitypic DENV NAb profiles (OR = 4.05, 95% CI: 1.18 to 13.87). Among those with JEV NAbs, the association with symptomatic illness did not vary by antibody titer.

Interpretation

The prior existence of JEV NAbs was associated with an increased probability of symptomatic as compared to asymptomatic DENV illness. These findings are in contrast to previous studies suggesting an attenuating effect of heterologous flavivirus immunity on DENV disease severity.     

Assessment of genetic diversity in the sorghum reference set using EST-SSR markers

Selection and use of genetically diverse genotypes are key factors in any crop breeding program to develop cultivars with a broad genetic base. Molecular markers play a major role in selecting diverse genotypes. In the present study, a reference set representing a wide range of sorghum genetic diversity was screened with 40 EST-SSR markers to validate both the use of these markers for genetic structure analyses and the population structure of this set. Grouping of accessions is identical in distance-based and model-based clustering methods. Genotypes were grouped primarily based on race within the geographic origins. Accessions derived from the African continent contributed 88.6 % of alleles confirming the African origin of sorghum. In total, 360 alleles were detected in the reference set with an average of 9 alleles per marker. The average PIC value was 0.5230 with a range of 0.1379–0.9483. Sub-race, guinea margaritiferum (Gma) from West Africa formed a separate cluster in close proximity to wild accessions suggesting that the Gma group represents an independent domestication event. Guineas from India and Western Africa formed two distinct clusters. Accessions belongs to the kafir race formed the most homogeneous group as observed in earlier studies. This analysis suggests that the EST-SSR markers used in the present study have greater discriminating power than the genomic SSRs. Genetic variance within the subpopulations was very high (71.7 %) suggesting that the germplasm lines included in the set are more diverse. Thus, this reference set representing the global germplasm is an ideal material for the breeding community, serving as a community resource for trait-specific allele mining as well as genome-wide association mapping.     

Estimates of Dengue Force of Infection in Children in Colombo,Sri Lanka

Dengue is the most important vector-borne viral disease worldwide and a major cause of childhood fever burden in Sri Lanka, which has experienced a number of large epidemics in the past decade. Despite this, data on the burden and transmission of dengue virus in the Indian Subcontinent are lacking. As part of a longitudinal fever surveillance study, we conducted a dengue seroprevalence survey among children aged <12 years in Colombo, Sri Lanka. We used a catalytic model to estimate the risk of primary infection among seronegative children. Over 50% of children had IgG antibodies to dengue virus and seroprevalence increased with age. The risk of primary infection was 14.1% per year (95% CI: 12.7%–15.6%), indicating that among initially seronegative children, approximately 1 in 7 experience their first infection within 12 months. There was weak evidence to suggest that the force of primary infection could be lower for children aged 6 years and above. We estimate that there are approximately 30 primary dengue infections among children <12 years in the community for every case notified to national surveillance, although this ratio is closer to 100∶1 among infants. Dengue represents a considerable infection burden among children in urban Sri Lanka, with levels of transmission comparable to those in the more established epidemics of Southeast Asia.     

Propyl-2-(8-(3,4-Difluorobenzyl)-2′,5′-Dioxo-8-Azaspiro[Bicyclo[3.2.1] Octane-3,4′-Imidazolidine]-1′-yl) Acetate Induces Apoptosis in Human Leukemia Cells through Mitochondrial Pathway following Cell Cycle Arrest

Background

Due to the functional defects in apoptosis signaling molecules or deficient activation of apoptosis pathways, leukemia has become an aggressive disease with poor prognosis. Although the majority of leukemia patients initially respond to chemotherapy, relapse is still the leading cause of death. Hence targeting apoptosis pathway would be a promising strategy for the improved treatment of leukemia. Hydantoin derivatives possess a wide range of important biological and pharmacological properties including anticancer properties. Here we investigated the antileukemic activity and mechanism of action of one of the potent azaspiro hydantoin derivative, (ASHD).

Materials and Methods

To investigate the antileukemic efficacy of ASHD, we have used MTT assay, cell cycle analysis by FACS, tritiated thymidine incorporation assay, Annexin V staining, JC1 staining and western blot analysis.

Results

Results showed that ASHD was approximately 3-fold more potent than the parent compounds in inducing cytotoxicity. Tritiated thymidine assay in conjunction with cell cycle analysis suggests that ASHD inhibited the growth of leukemic cells. The limited effect of ASHD on cell viability of normal cells indicated that it may be specifically directed to cancer cells. Translocation of phosphatidyl serine, activation of caspase 3, caspase 9, PARP, alteration in the ratio of BCL2/BAD protein expression as well as the loss of mitochondrial membrane potential suggests activation of the intrinsic pathway of apoptosis.

Conclusion

These results could facilitate the future development of novel hydantoin derivatives as chemotherapeutic agents for leukemia.